Biosensors, biocompatibilization of materials, and surface science require the investigation of the sorption processes and the 3-D organization of proteins at solid/liquid interface.

Many qualitative methods but few quantitative methods and even fewer real-time measurements ! Laurent FRANCIS – Biosensors 2004

http://www.aad.org/education/CollagenFigure1.htm

Collagen

Fibrinogen

Fibrillar protein of the extracellular matrix

Blood protein

Triple helix 300 nm long 1.5 nm diameter Weight ~ 300 kDa

Three globular domains linked by fibrillar segments Weight ~ 340 kDa

Forms elastic fibers

Causes blood clotting

How to investigate in liquids the behavior of adsorbed proteins ? Laurent FRANCIS – Biosensors 2004

A novel biosensor platform integrating Love mode acoustic wave and surface plasmon resonance for the investigation of protein layers Laurent FRANCIS*1,2, Jean-Michel FRIEDT3, Carmen BARTIC2, Patrick BERTRAND1, Andrew CAMPITELLI2 1 PCPM,

Université catholique de Louvain, Belgium 2 Biosensors Group, IMEC, Belgium 3 LMN, Université de Franche-Comté, France

Biosensors 2004 – Grenada, Spain – May 26th, 2004 Laurent FRANCIS – Biosensors 2004

OUTLINE „

Introduction

„

Love mode surface acoustic wave (SAW) Structure and characteristics „ Sensitivity enhancement „

„

Surface plasmon resonance (SPR)

„

Combined SAW/SPR Technique „ Method „ Application to collagen and fibrinogen „

„Conclusions

Laurent FRANCIS – Biosensors 2004

SURFACE ACOUSTIC WAVE

SAW

SURFACE PLASMON RESONANCE

SPR

Label-free sensing on a common surface increased system integration fast analysis low analyte volume identical event simplicity of sample preparation Real time measurement adsorption kinetic dynamic evolution of the layer Laurent FRANCIS – Biosensors 2004

LOVE MODE SAW SENSOR: STRUCTURE Overcoated SAW delay line is a mass sensitive device. Shear horizontal polarized acoustic wave that results in low loss with liquid loading. Substrates: α-SiO2, LiNbO3, LiTaO3 Guiding layers: SiO2, polymers/epoxies, ZnO Delay line structure

Experimental transfer function h

I.L.

Phase

φ

Laurent FRANCIS – Biosensors 2004

LOVE MODE SAW SENSOR: CHARACTERISTICS The dispersion of Love mode is ruled by - the acoustic velocity V2 = µ / ρ - the acoustic impedance Z2 = ρ ∗ µ Mechanical properties are function of - the selected guiding materials; - the structure (processing parameters, i.e. PECVD).

Experimental phase velocity (m/s)

Laurent FRANCIS – Biosensors 2004

Normalized thickness h/λ

LOVE MODE SAW SENSOR: MASS SENSITIVITY Mass sensitivity

λ

∆φ ⋅ S= 360o D m

λ acoustic wavelength D sensing length m surface density

Strong dispersion = high sensitivity: - H-rich PECVD SiO2 (reduced stiffness); - Gold (low velocity). Gold (50 nm) PECVD SiO2 ST-cut Quartz

S (cm2/g) Solid line = simulation Dot = experimental Laurent FRANCIS – Biosensors 2004

Thickness SiO2 (µm) with λ = 40 µm

SURFACE PLASMON RESONANCE SENSOR Surface Plasmon Resonance (SPR) is an optical method where collective electromagnetic modes are excited by a light source at metal-dielectric interface. SPR is sensitive to the thickness d and the refractive index n of dielectric added layers above specific metals (gold, silver, …). Simulated SPR signal Buffer Gold Prism Laser

θ Light intensity (a.u.)

∆θ SPR Angle θ (degrees) Laurent FRANCIS – Biosensors 2004

COMBINED SAW/SPR: SET-UP

EC cell SAW

SPR

SAW

SPR

modified IBIS II SPR (IBIS Technologies BV)

ST-cut quartz substrate 1.2 µm H-rich SiO2 10 nm Ti/ 50 nm Au wavelength 40 µm SU8/glass IDT capping

670 nm laser light Kretschmann configuration Laurent FRANCIS – Biosensors 2004

Cu-calibrated mass sensitivity of –260 cm2/g

COMBINED SAW/SPR: EXPERIMENTAL EXAMPLE Collagen 30 µg/ml adsorption on hydrophobic surface

SAW Phase φ

∆φ

SPR Angle θ

∆θ

Time (s) Laurent FRANCIS – Biosensors 2004

SAW/SPR MODELING

Au sensing surf. SAW

SPR

d = thickness of the layer (protein + water) d x = proportion of proteins in the layer ρ = density n = refractive index

Linear combination of the protein and the water: density refractive index

ρ ( x ) = x ρ PROTEIN + (1 − x ) ρWATER n( x ) = x nPROTEIN + (1 − x ) nWATER

The SAW shift ∆φ gives the surface density m of the layer:

  λ m=  ∆φ = ρ ( x ) d o  360 DS  The SPR shift ∆θ is simulated from d and n(x). Laurent FRANCIS – Biosensors 2004

SAW/SPR MODELING

x 100%

x 50%

SPR

x 10%

∆θ

d 1) Simulated SPR shifts ∆θ (d,x) 2) Experimental SPR shift 3) Allowed values for SAW shifts ∆φ (d,x) 4) Extraction of thickness d and protein content % Laurent FRANCIS – Biosensors 2004

EXPERIMENTAL RESULTS In-situ measurements of collagen and fibrinogen adsorbed on hydrophobic surfaces:

STRONG HYDRAT.

STACKED LAYERS

“DRY” LAYERS FOR SAW CALIB.

Analyte (µg/ml) Collagen 30 µg/ml Collagen 300 µg/ml Fibrinogen 46 µg/ml Fibrinogen 460 µg/ml

Surface density m (ng/cm2)

Thickness d (nm)

Protein content x (%)

1750 ± 150

16 ± 3

25 ± 15

2100 ± 200

19 ± 3

35 ± 10

750 ± 100

6 ± 1.5

50 ± 10

1500 ± 500

13 ± 2

50 ± 10

S-layer

560 ± 20

4.7 ± 0.7

75 ± 15

CTAB

135 ± 15

1 ± 0.1

100

Laurent FRANCIS – Biosensors 2004

EXPERIMENTAL RESULTS: ADSORPTION KINETICS

Normalized SAW

Normalized SPR The kinetic of the adsorption monitored by the two techniques is different: - wrong model ? Viscous effects neglected in the SAW ! - mass over/underestimation. Laurent FRANCIS – Biosensors 2004

DISCUSSION: DRAWBACKS OF THE TECHNIQUES SAW drawbacks - high temperature sensitivity - rigid mass and viscous effects combined - signal distortion due to interferences - different phase and group velocities SPR drawbacks - temperature dependence - signal distortion due to birefringence - optical interferences for multiple layers - data extraction through modeling - limited to metal surfaces (gold, silver) Combined technique - no interferences between SAW and SPR - complementary information allowing extraction of coupled parameters (ρ, n → x, d) on a same layer

Laurent FRANCIS – Biosensors 2004

DISCUSSION: LOVE MODE SAW MATERIAL COMBINATIONS SPR angle is function of n and of the light wavelength. Interferences and total internal reflection effects must be considered for a stack of layers. SPR sensitivity higher for θ closer to 90°. Conductive layers influence the SAW transfer function by shortcutting the transducers.

Light intensity (a.u.)

Laurent FRANCIS – Biosensors 2004

SPR Angle θ (degrees)

CONCLUSIONS „

Combined SAW/SPR technique: „ provides

information about the thickness and water content of protein layers;

„ real

time measurements, with some insights into the adsorption kinetics;

„ label-free

acoustic and optical method.

„

Ways to increase the Love mode SAW mass sensitivity: „ H-rich PECVD SiO2 „ Gold

„

Experimental results on collagen and fibrinogen adsorption demonstrate the potential and the limits of the combined technique.

Laurent FRANCIS – Biosensors 2004

ACKNOWLEDGEMENTS R. Giust (LOPMD, Université de Franche-Comté, France) for SPR simulation routines; M. Sára (Center for NanoBiotechnology, University of Vienna, Austria) for the S-layers; F.R.I.A. (Belgium) for financial support.

Slides of this presentation available at URL http://friedtj.free.fr/chua/biosensors2004.pdf or by e-mail : [email protected] Laurent FRANCIS – Biosensors 2004